It is now well-known that the majority of natural product chemical diversity has not been explored using traditional, cultivation-dependent strategies. Furthermore, a significant fraction of gene clusters from cultivated microbial sources remain silent under standard fermentation conditions. In theory, the heterologous expression of natural product gene clusters can provide access to chemical diversity from both uncultivated organisms and silent pathways. Recent studies have shown, however, that a major limitation of this approach is a lack of sufficient metabolic flux toward secondary metabolic pathways in an unengineered heterologous chassis, which limits production yield, making it challenging to dereplicate and identify heterologously produced natural products. To address these limitations we propose to develop a panel of robust heterologous production hosts to enable large- scale, systematic de-replication and discovery of natural products. This system will also allow for rapid, drop-in optimization of manufacturing and product purification for existing natural products, playing an important role for commercialization. In contrast to more widely-used biomanufacturing hosts, such as Escherichia coli and Saccharomyces cerevisiae, the engineered actinomycetes in this application produce complex natural products natively and contain the necessary secondary metabolic precursors, chaperone mechanisms for large biosynthetic protein folding, transcriptional activation machinery, and endogenous resistance mechanisms for the production of complex natural products. This Phase I application aims to develop a panel of optimized actinomycete hosts for heterologous production of natural products that, together, provide a tool for the consistent production of a broad spectrum of natural product classes regardless of their native expression levels. These actinomycete strains, chosen for their ability to produce diverse classes of natural products at high titer, will first b rationally engineered to remove competing natural product pathways to increase the availability of important precursors and targeted mutations to the ribosome will be added. These strategies have been shown to be a successful in developing Streptomyces coelicolor into an improved heterologous host. By generating a panel of these superhosts, we will broaden the spectrum of natural product clusters that can utilize this technology. In addition, we will build upon this panl of hosts using a novel microfluidic strain improvement technology. This microfluidic droplet cultivation and screening platform allows for a >10,000x improvement in the speed in colony screening and will be used to identify causal mutations that further increase the natural product production yield in these optimized hosts. Overall, this application aims to develop a panel of optimized production strains that will allow for more systematic, de-replication and discovery of natural products from uncultivated and genetically silent sources. In doing so, they will provide a renewable source of biologically-active chemical diversity from natural products.